In fusion

A genomic fragment of TPF2, including promoter until the codon before the stop, was amplified from genomic DNA and cloned into a pENTR/D plasmid by InFusion (Takara) to generate pENTR-gTPF2. The TPF2 fragment was transferred by LR reaction into a modified pEarleyGate302 that contains a 3xFLAG tag downstream of the gateway cassette to generate pEG-gTPF2-3xFLAG. This construct was transformed into Agrobacterium tumefaciens strain GV3101 C58C1, which was used to transform the tpf2-1 mutant by the floral dip method. A genomic fragment of MINU2, including promoter until the codon before the stop, and with an inserted XhoI site right upstream of the start codon was amplified from genomic DNA in two separate PCR reactions. These fragments were combined to amplify the full genomic sequence that was cloned into a pENTR/D plasmid by InFusion (Takara) to generate pENTR-gMINU2. A 3xFLAG tag was amplified and cloned into the unique XhoI site of pENTR-gMINU2. The 3xFLAG-gMINU2 fragment was transferred by LR reaction into a pMDC123 (51 (link)) to generate pMDC123-3xFLAG-gMINU2. This construct was transformed into Agrobacterium tumefaciens strain GV3101 C58C1, which was used to transform the minu2-1 mutant by the floral dip method.

A 1.2 kB region of the p230p gene (PF3D7_0208900) was amplified from PfMev genomic DNA using primers P230p.HA.F and P230p.HA.R (Appendix Table S2). These primers were designed to contain ˜15 bp overhangs which allowed insertion of the amplicon into the NgoMIV site of pRS (Swift et al,2020b (link)) by ligation independent cloning (In‐Fusion, Clontech). The plasmid was digested with BglII to excise a 210bp fragment between two BglII sites in the p230p gene. The excised region was replaced with an oligo comprised of complementary primers attBr. InF.F and attBr. InF.R (Appendix Table S2) using In‐Fusion to generate an attB site flanked by p230p homology arms. The plasmid was then digested with BsaI to insert a segment of DNA encoding a guide RNA targeting the excised region of p230p. Complementary primers P230p.gRNA.F and P230p.gRNA.R (Appendix Table S2) were annealed and inserted using ligation independent cloning with In‐Fusion (Clontech). The resulting plasmid pRS‐P230p was used along with pCasG (Rajaram et al,2020 (link)) in transfections for markerless insertion of the attB element into the P230p locus of PfMev parasites. After 48 h, transfectants were selected with 1.5 μM DSM1 for 7 days and 2.5 nM WR99210 for 10 days. Parasite clones were characterized by genotyping PCRs (Fig 2A) using primers described in Fig EV1 and Appendix Table S2.